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J. Biol. Chem., Vol. 277, Issue 35, 31863-31870, August 30, 2002

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Lipopolysaccharide Down-regulates Sp1 Binding Activity by Promoting Sp1 Protein Dephosphorylation and Degradation^*

Xiaobing Ye and Shu Fang Liu

From the Department of Medicine, Division of Pulmonary and Critical Care Medicine, Long Island Jewish Medical Center, Long Island Campus for the Albert Einstein College of Medicine, New Hyde Park, New York 11040

Received for publication, June 4, 2002, and in revised form, June 26, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We examined the in vivo effect of lipopolysaccharide (LPS) on Sp1 (promoter-selective transcription factor 1) DNA binding activity and studied the mechanisms involved in mouse lungs. The Sp1 DNA complex displayed a major band composed of Sp1, Sp2, and Sp3 trimer and a minor band composed of Sp3 homodimer. Compared with control, nuclear proteins from lungs challenged with LPS for 60, 90, 120, 150, 180, and 240 min, respectively, showed a markedly reduced Sp1 binding activity. Down-regulation of Sp1 binding activity was accompanied by a reduced expression of two Sp1-dependent genes (endothelial nitric oxide synthase and cyclooxygenase-1). Immunoprecipitation-Western blot experiments demonstrated that LPS dephosphorylated Sp1 protein at serine and threonine residues but not at the tyrosine residue. Dephosphorylation of Sp1 protein in vitro significantly reduced Sp1 DNA binding activity. Deglycosylation of Sp1 protein also reduced Sp1 binding activity. However, LPS did not cause Sp1 deglycosylation. LPS markedly reduced nuclear Sp1 protein level but had no significant effect on Sp1 mRNA abundance and on Sp1 protein nuclear translocation. Both Sp1 protein dephosphorylation and Sp1 protein degradation are temporally correlated to the reduced Sp1 binding activity. Our results demonstrate that challenge of mice with LPS in vivo down-regulates Sp1 DNA binding activity through promoting Sp1 protein dephosphorylation and degradation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Lipopolysaccharide (LPS)¹ is a principle mediator of sepsis, septic shock, and other inflammatory disorders (1). Exposure of host cells to LPS triggers the release a battery of host defense molecules including cytokines, chemokines, interferons, cell adhesion molecules, lipid mediators, reactive oxygen species, and nitrogen intermediates. Although these molecules are essential to host defense against bacterial infection, excessive production of these mediators can cause circulatory collapse and organ damage, leading to septic shock syndrome, a condition that results in ~20,000 annual deaths in the United States (2). Cellular activation by LPS is mediated by signal transduction cascade initiated by the interaction between LPS and its receptor, CD14 (3). CD14-mediated recognition and signaling require four proteins: LPS-binding protein (4, 5), CD14 (4-6), MD-2 (7), and Toll-like receptor family proteins (8, 9). Several intracellular signaling cascades are involved in the LPS signaling, including the Ras-Raf-mitogen-activated protein kinase pathway (10), protein kinase C pathway (11), ceramide pathway (12), phosphatidylinositol 3-kinase pathway (13), and the stress-activated protein kinase pathway (14). Molecules involved in cytokine receptor signaling also play important roles in the LPS signaling process (15-17). Thus, LPS can stimulate multiple signal pathways, leading to a diversity of biological effects.

Transcription factors are usually the final target of many signal transduction cascades that lead to gene transcription and may play important roles in the LPS signaling. Transcription factors known to mediate LPS response include NF-B, activating protein-1, and nuclear factor interleukin-6 (18). The roles of other transcription factors in LPS signaling remain to be studied. One transcription factor that may be a target of LPS signaling is the promoter-selective transcription factor (Sp1).

The GC box-binding protein, Sp1, is a ubiquitous transcription factor that belongs to the Sp family of transcription factors, consisting of Sp1, Sp2, Sp3, and Sp4 (19). Sp1 has been implicated in the transcription of large number of genes (for a complete list, check on PubMed, using key word sp1), particularly housekeeping genes, tissue-specific genes, and genes involved in growth regulation (19-21). Sp1 activities are regulated by a variety of stimuli. Most of these regulations occur through either post-translational modification or alteration of Sp1 protein abundance. The principal known post-translational modifications are phosphorylation and glycosylation through the O-linkage of the monosaccharide, N-acetylglucosamine (O-GlcNAc) (22). Depending on cell type and stimuli, phosphorylation of Sp1 protein can increase (23), decrease (24-26), or have no effect on Sp1 activity (27). Reduced O-GlcNAcylation of Sp1 protein results in a decreased Sp1 binding activity (28). However, increased O-GlcNAcylation of Sp1 protein can regulate Sp1 activity positively (28, 29) or negatively (30). O-GlcNAcylation of Sp1 protein inhibits its interaction with TATA-binding protein-associated factor, TAFI 10, or holo-Sp1 (31). The abundance of cellular Sp1 protein is principally regulated via proteolytic activity, although the proteases involved are quite variable with cell types and stimuli (26, 28, 32, 33). On rat pituitary adenoma cells, epidermal growth factor and okadaic acid cause Sp1 degradation via a cysteine protease (26), whereas in normal rat kidney cells, glucose starvation and adenylate cyclase activation stimulate Sp1 proteolysis through a 26 S proteasome system (28). In mouse T cells, EL-4 cells, and in human T cells, Jurkat cells, retinoid promotes Sp1, proteolysis by a mechanism that involves activation of caspase (32). In the green monkey kidney cell line, CV-1, Sp1 protein is degraded by a cathepsin-like cysteine protease under unstimulated conditions (33).

Although we have previously reported that challenge of rat with LPS down-regulates Sp1-DNA binding activity in the lungs (34), the mechanisms regulating Sp1 activity during sepsis and the biological significance of this down-regulation remain to be elucidated. The composition of the Sp1-DNA binding complex under in vivo conditions has not been determined. Additionally, it is not known whether this down-regulation is a universal phenomenon. In this study, we have characterized the time course profile of LPS-induced down-regulation of Sp1 binding activity in the mouse lungs, determined the composition of the Sp1-DNA binding complex, elucidated the mechanisms mediating the LPS-induced down-regulation of Sp1 binding activity, and studied the biological significance of this down-regulation. Our data demonstrated that LPS down-regulates Sp1 activity through promoting Sp1 protein dephosphorylation and degradation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Animal Protocol-- Adult FVB mice (25-30 g, body weight) were used in all studies. The institutional Animal Care Committee has proven all the animal protocols. Mice in control and LPS groups were injected with saline (1 ml/kg, intraperitoneally) or Salmonella enteritidis lipopolysaccharide (10 mg/kg, intraperitoneally), respectively. At 5, 10, 15, 30, 60, 90, 120, 150, 180, and 240 min after LPS injection, animals were killed by exsanguination. Lungs were removed, snap-frozen in liquid nitrogen, and used for extracting cytoplasmic and nuclear proteins.

Electrophoretic Mobility Shift Assay (EMSA)-- Nuclear and cytoplasmic proteins were extracted from mouse lungs as previously described (35). Sp1 consensus oligonucleotide probe (5'-ATTCGATCGGGGCGGGGCCAG-3') was end-labeled with [-³²P]ATP (Amersham Biosciences). Nuclear protein (10 µg) was incubated with 50,000 cpm ³²P-labeled Sp1 consensus oligonucleotide for 30 min in binding buffer consisting of 10 mM Tris-Cl, pH 7.5, 1 mM MgCl₂, 50 mM NaCl, 0.5 mM dithiothreitol, 0.5 mM EDTA, 4% glycerol, and 1 µg of poly(dI·dC) (Amersham Biosciences). The specificity of the Sp1 DNA binding was determined in competition reactions in which a 50-fold molar excess of unlabeled Sp1 oligonucleotide was added to the binding reaction 10 min prior to the addition of radiolabeled probe. In the supershift assay, antibody (1 µg) reactive to mouse Sp1, Sp2, Sp3, and Sp4 protein (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added to the reaction mixture immediately after the addition of radiolabeled Sp1 probe. Reaction was subjected to nondenaturing 4% polyacrylamide gel electrophoresis. Gel was vacuum-dried and exposed to x-ray film.

Western Blot Analysis-- Nuclear and cytoplasmic proteins were extracted from lungs of control mice and mice challenged with LPS for 5, 10, 15, 30, 60, 120, and 240 min, respectively. Equal amounts of cytoplasmic or nuclear proteins (20 µg/lane) were separated on 10% SDS-polyacrylamide gel under denaturing conditions. Proteins were electroblotted to nitrocellulose membrane (Bio-Rad). After incubation in blocking solution (5% dry milk in TBST) at room temperature for 2 h, the membrane was incubated with primary antibodies (Sp1 etc.) at room temperature for 1 h. The membrane was washed and incubated with secondary antibody conjugated to horseradish peroxidase at room temperature for 1 h. Peroxidase labeling was detected using the ECL Western blotting detection system (Amersham Biosciences).

Northern Blot Analysis-- The rat cyclooxygenase-1 (COX-1) (308 bp), GAPDH (309 bp), and mouse Sp1 (461 bp) cDNA probes were generated using reverse transcriptase-PCR using primers corresponding to published rat COX-1 (36) and GAPDH (37) and mouse Sp1 (GenBank^TM accession number NM-013672) cDNA sequences. Authenticities of PCR product were confirmed by dideoxy chain termination sequencing. The endothelial nitric-oxide synthase (eNOS) probe was full-length bovine eNOS cDNA probe (38). Total and poly(A)⁺ mRNA was isolated as previously described (35). Twenty µg of total RNA or 2 µg of mRNA from each sample was separated on a 1.2% denaturing agarose gel and transferred onto Biodyne nylon membrane (Pall Gelman Laboratory, Ann Arbor, MI). The membrane was incubated at 42 °C for at least 4 h in ULTRAhyb buffer (Ambion, Austin, TX). Membrane was hybridized with 0.5-2 × 10⁶ cpm/ml ³²P-labeled probes at 42 °C for 12-14 h. Blots were washed sequentially at decreasing concentrations of SSC plus 0.1% SDS and increasing temperature (final wash, 0.1× SSC plus 0.1% SDS at 60 °C for 30 min) and exposed to x-ray film.

Nuclear Protein Dephosphorylation-- Nuclear protein (10 µg) from control lungs was incubated with 5 units of calf intestinal alkaline phosphatase (CIP; New England Biolabs) for 0.5 h at 37 °C in dephosphorylation buffer (25 mM HEPES, pH 7.5, 34 mM KCl, 50 mM MgCl₂). The reaction was stopped by the addition of a mixture of phosphatase inhibitors to a final concentration of 10 mM sodium fluoride, 10 mM sodium vanadate, 10 mM potassium pyrophosphate, and 5 mM sodium phosphate. The dephosphorylated nuclear protein (NP) was used for EMSA. We carried out three groups of reactions: control, NP alone; CIP, NP, buffer, and CIP; and mock, NP plus dephosphorylation buffer without CIP. To verify that treatment of control nuclear proteins with CIP dephosphorylates Sp1 protein, 150 µg of control and CIP- and mock-treated NP was immunoprecipitated with Sp1 antibody and subjected to Western blot using antibody against phosphoserine and Sp1.

Nuclear Protein Deglycosylation-- Nuclear protein (10 µg) from control lungs was incubated with 0.1 units of -N-acetylglucosaminidase (NAG; Sigma) in reaction buffer (50 mM citric acid, 100 mM NaCl, and 0.01% bovine serum albumin, pH 5) at 25 °C for 20 min. The reaction was stopped by the addition of 5 µl of 600 mM borate buffer. The deglycosylated nuclear proteins were used for EMSA. We carried out three groups of reactions: control (NP alone); NAG, NP, buffer, and NAG (0.1 units); and mock (NP plus deglycosylation buffer without NAG). To verify that treatment of control nuclear proteins with NAG deglycosylates Sp1 protein, 150 µg of control and NAG- and mock-treated NP was immunoprecipitated with Sp1 antibody and subjected to Western blot using antibody against O-linked acetylglucosamine and Sp1.

Immunoprecipitation-- Nuclear proteins (350 µg) from control lungs and lungs challenged with LPS for 15, 30, 60, 120, and 240 min, respectively, were immunoprecipitated with 4 µg of Sp1 antibody and 20 µl of protein G Plus-agarose (Santa Cruz Biotechnology, Inc.) in binding buffer overnight at 4 °C. The Sp1-antibody-protein G complexes were centrifuged at 1000 × g, and the pellets were washed five times with binding buffer. The immunoprecipitation-Sp1 protein was resuspended in Western blot sample buffer, boiled, electrophoresed on SDS-PAGE, and transferred onto nitrocellulose membrane. The immunoblots were developed with antibodies against phosphoserine, phosphothreonine, phosphotyrosine (Zymed Laboratories), or O-linked acetylglucosamine (Affinity BioReagents).

Data Analysis and Statistics-- Bands on EMSA and Northern blot autoradiogram film were quantitated using a RS700 densitometer linked to a computer analysis system (Bio-Rad). The relative eNOS, COX-1, and Sp1 mRNA levels were normalized by their corresponding GAPDH bands. Statistical analysis of multiple comparison data was performed by one-way analysis of variance or Kruskal-Wallis Rank test. Comparison between two groups was analyzed using t test or the Mann-Whitney U test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

LPS Down-regulated Sp1 DNA Binding Activity in Mouse Lungs-- We have previously reported that LPS challenge in vivo down-regulated Sp1 DNA binding activity in rat lungs (34). To verify whether this is a universal phenomenon, we now characterize the LPS-induced down-regulation of Sp1 binding activity in the mouse lungs. We examine the time course profile of this down-regulation in more detail. Nuclear proteins were extracted from control lungs and lungs challenged with LPS for 5, 10, 15, 30, 60, 90, 120, 150, 180, and 240 min, respectively, and were tested for their ability to bind to Sp1 oligonucleotide probes in EMSA. Competition study showed that the Sp1-DNA binding complex was virtually completely displaced in the presence of a 50-fold molar excess of unlabeled Sp1 oligonucleotide probe (Fig. 1A, lane 3). In contrast, a 50-fold molar excess of unlabeled NF-B oligonucleotide probe had no effect on the Sp1-DNA binding complex (Fig. 1A, lane 4), indicating the specificity of Sp1 DNA binding. There were two bands in the Sp1-DNA complex. The major band was supershifted by Sp1, Sp2, and Sp3 antibodies either individually or in combination, indicating that it is composed of Sp1, Sp2, and Sp3 trimer (Fig. 1B). However, Sp2 appears to constitute only a small portion of the major band, since a combination of Sp1 and Sp3 antibodies caused a significantly greater supershift than that caused by the combination of Sp1 and Sp2 antibodies (Fig. 1B). The minor band was supershifted by Sp3 antibody only, indicating that it is composed of Sp3/Sp3 homodimer (Fig. 1B). Sp4 antibody did not supershift either band. The Sp1 DNA binding activity was strong in nuclear proteins from control lungs but was markedly reduced in nuclear proteins from lungs challenged with LPS for 60, 120, and 240 min (Fig. 2A). We quantified the Sp1 band intensity using densitometry and expressed as arbitrary optical density units. Compared with controls, LPS caused a 79, 91, and 54% reduction in Sp1 band intensity at 60, 120, and 240 min post-LPS (Fig. 2B). To obtain a more detailed time course profile of this Sp1 down-regulation, we examined Sp1 DNA binding activity in nuclear protein from lungs challenged with LPS for 90, 150, and 180 min, respectively. As illustrated in Fig. 2C, the Sp1 band intensity at 90 and 150 min post-LPS was similar to that at 120 min post-LPS (Fig. 2A, lane 7). The band intensity increased slightly at 180 min post-LPS but was still less intense compared with that at 240 min (Fig. 2A, lane 8, versus Fig. 2C, lane 4). Thus, challenge of mice with LPS in vivo down-regulated Sp1 DNA binding activity in the lungs. This down-regulation was seen at 60 min, maximized at 120-150 min, and gradually recovered from 180 min onward.

View larger version (59K): [in this window] [in a new window]   Fig. 1.   Autoradiogram of EMSA showing the characteristics of Sp1-DNA complex in the mouse lungs. A, competition study. Lane 1, Sp1 probe without nuclear extract; lane 2, nuclear extract from control lungs; lanes 3 and 4, the same sample as in lane 2 but including a 50-fold molar excess of unlabeled Sp1 probe (lane 3) or NF-B probe (lane 4). B, supershift assay using nuclear extract from control lungs. Sp1 DNA binding reaction was carried out in the absence (control; Con) and presence of antibodies to Sp1 (Sp1), Sp2 (Sp2), Sp3 (Sp3), Sp4 (Sp4) or a combination of Sp1 and Sp2 (Sp1 + 2), Sp1 and Sp3 (Sp1 + 3), or Sp1, Sp2, and Sp3 (Sp1 + 2 + 3) antibodies. A shift was observed with Sp1, Sp2, and Sp3 but not Sp4 antibodies. Note that the combination of Sp1 plus Sp3 or Sp1 plus Sp2 plus Sp3 virtually fully shifted this complex.

View larger version (56K): [in this window] [in a new window]   Fig. 2.   Time course of LPS-induced down-regulation of Sp1 binding activity in mouse lungs. Mice were challenged with LPS (10 mg/kg, intraperitoneally) for the periods of time indicated. Nuclear protein was extracted from lungs of control (Con) and lungs challenged with LPS and was subjected to EMSA. A, autoradiogram of EMSA showing the time course of LPS-induced down-regulation of Sp1 binding activity. B, Sp1 band intensity was quantified using densitometry and expressed as arbitrary optical density units. Con, control. Values shown are the mean ± S.E. of five animals in each group. C, autoradiogram of EMSA showing the effects of LPS challenge for 90, 150, and 180 min, respectively, on Sp1 binding activity in the lungs.

LPS Down-regulated eNOS and COX-1 Gene Expression-- To determine the biological significance of the LPS-induced down-regulation of Sp1 activity, we examined the effect of LPS on the gene expressions of eNOS and COX-1, two of the most important Sp1-dependent genes (39, 40), in rat lungs challenged with LPS for 4 h. This is the time point 2 h after the maximum reduction in Sp1 DNA binding activity was observed. Due to the small size, it was impossible to perform EMSA, Western blot, and Northern blot on the same mouse lung. To see a better correlation between Sp1 activity and eNOS or COX-1 gene expression, we carried out Northern blot using rat lungs challenged with LPS for 4 h. We have previously showed a down-regulated Sp1 activity in these lungs (34). Using eNOS- and COX-1-specific probes, we detected a single eNOS band of ~4.3 kb and a single COX-1 band of ~2.8 kb in RNA from control lungs (Fig. 3). Both eNOS and COX-1 mRNA band intensity was greatly reduced in RNA from lungs challenged with LPS for 4 h (Fig. 3). Thus, down-regulation of Sp1 binding activity by LPS is accompanied by and correlated to a down-regulation of the expression of Sp1-dependent genes.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Northern blot showing LPS-induced down-regulation of eNOS and COX-1 mRNA expression in the lungs. GAPDH mRNA serves as internal controls. Two µg of mRNA was separated on agarose gel, transferred to nylon membrane, and subjected to Northern blot. A, Northern blot autoradiogram showing LPS-induced down-regulation of eNOS and COX-1 mRNA expression. B, the eNOS and COX-1 band intensity was quantified using densitometry and expressed as eNOS/GAPDH or COX-1/GAPDH ratio. Con, control. Values shown are the mean ± S.E. of three animals in each group.

LPS-dephosphorylated Sp1 Protein-- We next examined the mechanism of LPS-induced down-regulation of Sp1 binding activity. We first want to know whether LPS causes Sp1 protein phosphorylation or dephosphorylation and, if so, which residue is phosphorylated or dephosphorylated. We immunoprecipitated Sp1 protein from nuclear extract of control lungs and lungs challenged with LPS for 15, 30, 60, 120, and 240 min, respectively. The immunoprecipitation-Sp1 proteins were immunoblotted to antibodies against phosphoserine, phosphothreonine, and phosphotyrosine. As illustrated in Fig. 4, anti-phosphoserine and anti-phosphothreonine antibodies detected a strong band, and anti-phosphotyrosine antibody detected no band on control Sp1 protein, indicating that Sp1 protein is phosphorylated at serine and threonine residues but not at tyrosine residue under normal physiological conditions. LPS reduced the phosphoserine and phosphothreonine band intensity in a time-dependent manner (Fig. 4). Anti-phosphotyrosine antibody detected a light band on Sp1 proteins from lungs challenged with LPS for 15, 30, 60, and 120 min, respectively (Fig. 4). This phosphotyrosine band became undetectable on Sp1 protein from lungs challenged with LPS for 240 min, when the phosphoserine and phosphothreonine bands reappeared (Fig. 4). These results indicate that LPS caused Sp1 protein dephosphorylation at serine and threonine residues and Sp1 protein phosphorylation at tyrosine residue. At 120 min post-LPS, phosphoserine and phosphothreonine bands became virtually undetectable (Fig. 4), suggesting complete dephosphorylation. This pattern of changes in Sp1 phosphorylation state was temporally correlated to the pattern of changes in LPS-induced reduction of Sp1 DNA binding activity (Figs. 2 and 4).

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Western blot photograph showing LPS-induced dephosphorylation of Sp1 protein. Nuclear protein was extracted from control lungs (Con) and lungs challenged with LPS for 15, 30, 60, 120, and 240 min, respectively. Sp1 protein was immunoprecipitated with anti-Sp1 antibody and immunoblotted to antibodies against Sp1 (Sp1), phosphoserine (serine), phosphothreonine (threonine), and phosphotyrosine (tyrosine).

Sp1 Protein Dephosphorylation Reduced Its DNA Binding Activity-- To establish a link between LPS-induced Sp1 protein dephosphorylation and reduced Sp1 binding activity, we incubated nuclear proteins from control lungs with calf intestinal alkaline phosphatase to dephosphorylate Sp1 protein and tested the ability of the dephosphorylated proteins to bind to Sp1-specific oligonucleotide probe. Compared with control, dephosphorylated nuclear proteins showed a dramatically reduced Sp1 DNA binding activity (Fig. 5A, CIP). By contrast, mock dephosphorylation had no effect on Sp1 DNA binding activity (Fig. 5A). The reduced Sp1 DNA binding activity seen in CIP-treated nuclear protein (Fig. 5A, CIP) was not a result of reduced Sp1 protein level (Fig. 5B, CIP) but rather a result of Sp1 protein dephosphorylation (Fig. 5B, CIP). These results established a link between Sp1 protein dephosphorylation and the reduced Sp1 DNA binding activity.

View larger version (70K): [in this window] [in a new window]   Fig. 5.   Dephosphorylation of Sp1 protein reduced its DNA binding activity. NP from control lungs was incubated with CIP (0.5 units/µg of NP). The dephosphorylated NP was used for EMSA or subjected to immunoprecipitation-Western blot. Con, NP alone; CIP, NP, buffer, and CIP; Mock, NP plus dephosphorylation buffer without CIP. A, autoradiogram of EMSA showing dephosphorylation of control NP reduced Sp1 DNA binding activity. B, Western blot showing that treatment of control NP with CIP dephosphorylated Sp1 protein but had no effect on Sp1 protein level.

Deglycosylation of Sp1 Protein Reduced Its DNA Binding Activity-- To test whether change in Sp1 protein glycosylation state also affects Sp1 binding activity, the O-linked N-acetylglucosamine was removed from control Sp1 protein by incubating nuclear proteins from control lungs with NAG. The deglycosylated nuclear protein was tested for its ability to bind to Sp1-specific oligonucleotide probe. The mock deglycosylation reaction contained all of the components without NAG. Mock deglycosylation of nuclear protein moderately reduced Sp1 binding activity, suggesting that deglycosylation buffer interferes with the Sp1 DNA binding reaction (Fig. 6A). However, deglycosylation of nuclear protein caused an over 80% reduction in Sp1 DNA binding activity (Fig. 6A, NAG). NAG treatment had no effect on Sp1 protein level (Fig. 6B, NAG) but caused Sp1 protein deglycosylation (Fig. 6B, NAG), indicating that NAG treatment reduces Sp1 DNA binding activity by causing Sp1 protein deglycosylation.

View larger version (75K): [in this window] [in a new window]   Fig. 6.   Deglycosylation of Sp1 protein reduced its DNA binding activity. NP from control lungs was incubated with NAG (0.1 units/10 µg of NP). The deglycosylated NP was used for EMSA or subjected to immunoprecipitation-Western blot. Con, NP alone; NAG, NP, buffer, and NAG; Mock, NP plus deglycosylation buffer without NAG. A, autoradiogram of EMSA showing deglycosylation of control NP reduced Sp1 DNA binding activity. B, Western blot showing that treatment of control NP with NAG deglycosylated Sp1 protein but had no effect on Sp1 protein level.

LPS Did Not Cause Sp1 Protein Deglycosylation-- Sp1 is a glycoprotein and is known to undergo glycosylation or deglycosylation under various physiological and pathophysiological conditions. We determined the effect of LPS on Sp1 glycosylation and linked the change in Sp1 glycosylation state to the reduced Sp1 DNA binding activity. Immunoprecipitated Sp1 protein from nuclear extract of control lungs and lungs challenged with LPS for 15, 30, 60, 120, and 240 min, respectively, was immunoblotted to antibody against O-linked N-acetylglucosamine. Fig. 7 displayed the time course of the LPS effect on Sp1 protein glycosylation. As expected, there was a high level of O-linked N-acetylglucosamine on control Sp1 protein. Sp1 proteins from LPS-challenged lungs showed a similar level of glycosylation (Fig. 7), indicating that Sp1 deglycosylation is not involved in the LPS-induced down-regulation of Sp1 binding activity.

View larger version (31K): [in this window] [in a new window]   Fig. 7.   Western blot photograph showing LPS had no effect on Sp1 protein glycosylation. NP was extracted from control lungs (Con) and lungs challenged with LPS for 15, 30, 60, 120, and 240 min, respectively. Sp1 protein was immunoprecipitated with anti-Sp1 antibody, and immunoblotted with antibody against Sp1 (Sp1) or O-linked acetylglucosamine (O-GlcNAc).

LPS Reduced Nuclear Sp1 Protein Level-- We next examined the effects of LPS on Sp1 protein level. We detected Sp1 protein in nuclear extract from control lungs and lungs challenged with LPS for 5, 10, 15, 30, 60, 120, and 240 min, respectively, using Western blot. As shown in Fig. 8A, there was a high level of Sp1 protein in control nuclear extract, which was not significantly affected by short periods of LPS challenge (shorter than 30 min). However, LPS challenge for 60 min or longer significantly reduced the Sp1 protein level (Fig. 8A). The Sp1 protein band became undetectable in nuclear protein from lungs challenged with LPS for 120 min and reappeared in nuclear extract from lungs challenged with LPS for 240 min (Fig. 8A). This pattern of changes in Sp1 protein level correlated well with the pattern of changes in Sp1 binding activity (Figs. 2A and 8A). The LPS-induced reduction in Sp1 protein level was not a result of universal protein degradation induced by LPS. Fast Green staining of the membrane used for Western blot in Fig. 8A showed an equal amount of total protein in every lane of the membrane (Fig. 8B).

View larger version (108K): [in this window] [in a new window]   Fig. 8.   Western blot photograph showing LPS promotes Sp1 protein degradation. NP was extracted from control lungs (Con) and lungs challenged with LPS for 5, 10, 15, 30, 60, 120, and 240 min, respectively. Twenty µg of NP was separated on SDS-PAGE, transferred onto nitrocellulose membrane. The membrane was stained with Fast Green dye and prepared for the Western blot procedure using antibody against Sp1 protein. A, Western blot photograph showing LPS-induced Sp1 protein degradation. Note the small size peptide reactive to the Sp1 antibody in NP of 60 and 240 min post-LPS. No Sp1 band was detected in NP of 120 min post-LPS, suggesting complete degradation. B, Fast Green staining of nitrocellulose membrane to confirm equal loading in each lane.

LPS Did Not Reduce Sp1 mRNA Abundance-- To ascertain whether the LPS-induced down-regulation of Sp1 protein level is a result of a decreased Sp1 RNA or protein synthesis or increased Sp1 protein degradation, we examined the LPS effect on Sp1 mRNA abundance using Northern blot analysis. Because Sp1 protein level did not show a reduction until 60 min post-LPS, we analyzed the Sp1 mRNA level only at three time points that precede the time point when the early Sp1 protein reduction was seen. Using the mouse Sp1 cDNA probe, we detected two Sp1 mRNA transcripts with approximate sizes of 8.2 and 4 kb, respectively. Human Sp1 mRNA has also been reported to have two transcripts (8.2 and 4.1 kb) (41). Sp1 mRNA abundance (both 8.2- and 4-kb transcripts) varied considerably between control lungs and also between LPS-challenged lungs. Overall, LPS had no significant effect on Sp1 mRNA level (Fig. 9).

View larger version (26K): [in this window] [in a new window]   Fig. 9.   Northern blot showing that LPS had no effect on Sp1 mRNA abundance. Total RNA was extracted from control lungs (Con) and lungs challenged with LPS for 15, 30, and 60 min, respectively. Twenty µg of total RNA was separated on denaturing gel, transferred to nylon membrane, and subjected to Northern blot analysis using Sp1- and GAPDH-specific probe. A, Northern blot autoradiogram showing the effects of LPS on the abundance of the two Sp1 mRNA transcripts (4 and 8 kb). B, the Sp1 band intensity was quantified using densitometry and expressed as Sp1/GAPDH ratio. Open bar, 4-kb Sp1 mRNA transcript; hatched bar, 8.2-kb Sp1 mRNA transcript. Con, control. Values shown are the mean ± S.E. of five animals in each group.

LPS Did Not Alter Sp1 Protein Nuclear Translocation-- LPS may alter the dynamics of Sp1 protein nuclear import. To test this possibility, we compared the Sp1 protein abundance in cytoplasmic and nuclear extracts from control lungs and lungs challenged with LPS. We reasoned that if LPS decreases nuclear import (or increases nuclear export) of Sp1 protein, LPS-challenged lungs that showed a greatly reduced Sp1 protein level in their nuclear extract should display a significantly increased Sp1 protein level in cytoplasmic extract (Sp1 protein plasmatic retention). As illustrated in Fig. 10, cytoplasmic extracts from lungs challenged with LPS for 60, 120, and 240 min, respectively, showed a similar or lesser Sp1 protein abundance compared with control, although these lungs had significantly reduced Sp1 protein level in nuclear extract (Fig. 10). Thus, LPS did not appear to alter Sp1 protein nuclear translocation.

View larger version (21K): [in this window] [in a new window]   Fig. 10.   Western blot photograph comparing Sp1 protein content in nuclear and cytoplasmic extracts from the same groups of lungs. LPS did not cause cytoplasmic retention of Sp1 protein.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have previously demonstrated that challenge of rat with LPS in vivo down-regulates Sp1 binding activity in rat lungs (34). Here we extend our previous study by elucidating the molecular mechanisms mediating the LPS effect and by examining the biological significance of this down-regulation in the mouse and rat lungs. We showed that challenge of mice with LPS dramatically down-regulated the Sp1 DNA binding activity in the lungs in a time-dependent manner. Down-regulation of Sp1 binding activity was accompanied by and temporally correlated to the down-regulation of the expression of eNOS and COX-1 genes, the two most important Sp1-dependent genes. The Sp1-DNA complex showed two bands, a major band composed of Sp1, Sp2, and Sp3 trimer and a minor band composed of Sp3/Sp3 homodimer. Several possible mechanisms have been examined, including the following: 1) LPS down-regulates Sp1 mRNA and protein expression; 2) LPS reduces nuclear translocation of Sp1 protein; 3) LPS causes Sp1 protein deglycosylation or dephosphorylation; and 4) LPS promotes Sp1 protein degradation. None of these possible mechanisms have previously been examined. LPS had no effect on Sp1 mRNA abundance, nor did it cause a cytoplasmic retention of Sp1 protein, indicating that LPS-induced down-regulation of Sp1 binding activity is unlikely to result from a decreased transcription of Sp1 gene or a reduced nuclear import of Sp1 protein. LPS caused Sp1 protein dephosphorylation at both serine and threonine residues but not tyrosine residue. Pretreatment of control NP with CIP in vitro dephosphorylated Sp1 protein and markedly reduced its DNA binding activity. Similarly, pretreatment of control NP with NAG in vitro had no effect on Sp1 protein level but deglycosylated Sp1 protein and reduced markedly Sp1 DNA binding activity. However, deglycosylation of Sp1 protein is unlikely to be involved in the LPS-induced down-regulation of Sp1 binding activity, since LPS challenge did not cause Sp1 protein deglycosylation. These results indicate that LPS reduces Sp1 binding activity through dephosphorylation of Sp1 protein and that dephosphorylation alone is sufficient to down-regulate Sp1 binding activity. We also showed that LPS reduced nuclear Sp1 protein content in a time-dependent manner and that this reduction temporally correlated with the down-regulation of Sp1 DNA binding activity, indicating that accelerated Sp1 protein degradation is another mechanism underlying the LPS-induced down-regulation of Sp1 binding activity. Thus, we demonstrated that challenge of mice with LPS in vivo down-regulated Sp1 binding activity in the lungs through two mechanisms, causing Sp1 protein dephosphorylation and promoting Sp1 protein degradation. Whether there is a causal relationship between Sp1 protein dephosphorylation and degradation is currently under investigation.

Phosphorylation is one of the most important post-translational modifications that alter protein function (42). Sp1 protein is known to be phosphorylated when cells are stimulated with various stimuli (23-26). However, our study is the first to show that Sp1 protein is phosphorylated under normal physiological conditions in vivo. We identified that Sp1 protein phosphorylation occurred at serine and threonine residues but not tyrosine residue in control lungs. LPS affected serine, threonine, and tyrosine phosphorylation differentially. LPS dephosphorylated Sp1 protein at serine and threonine residues but phosphorylated this protein at tyrosine residues. However, it is the dephosphorylation of serine and threonine but not phosphorylation of tyrosine that contributes to the reduced Sp1 DNA binding activity in response to LPS. Dephosphorylation of serine and threonine residues of Sp1 protein correlated well with the down-regulation of Sp1 DNA binding activity. Phosphoserine and phosphothreonine bands became undetectable at 60 and 120 min post-LPS when the Sp1 DNA binding activity was maximally reduced. Phosphoserine and phosphothreonine bands reappeared at 240 min post-LPS when Sp1 DNA binding activity partially recovered, although part of the reappearing phosphoserine and phosphothreonine bands may represent the increased Sp1 protein level. By contrast, phosphotyrosine band intensity increased when the Sp1 DNA binding decreased, and the phosphotyrosine band disappeared when the Sp1 DNA binding activity partially recovered at 240 min post-LPS. Our demonstration that in vitro treatment of control nuclear proteins with CIP dephosphorylated Sp1 protein and significantly reduced Sp1 DNA binding activity indicates that Sp1 protein phosphorylation is at least a partial prerequisite for Sp1 DNA binding. These data establish a linkage between LPS-induced Sp1 protein dephosphorylation and the reduced Sp1 DNA binding activity. Thus, LPS reduced Sp1 binding activity by dephosphorylating Sp1 protein under in vivo conditions. Our results are in line with the demonstration that cAMP-dependent protein kinase phosphorylates Sp1 protein and increases Sp1 DNA binding activities (23). However, our data contrast with three previous reports in which it was shown that phosphorylation of Sp1 protein induced by a DNA-dependent protein kinase, liver cell terminal differentiation, or stimulation with epidermal growth factor and okadaic acid, respectively, had no effect (27) or greatly reduced Sp1 DNA binding activity (24-26). It is not clear whether the difference between our result and the others is due to difference in cell types, difference in stimuli, or difference between in vivo and in vitro. Nevertheless, these results indicate that the effect of Sp1 phosphorylation on its function depends completely on cell types and stimuli.

Nuclear proteins are frequently modified by the addition of O-linked monosaccharide, O-GlcNAc (22). This modification undergoes dynamic changes in response to various signal transduction pathways, resulting in changes of its function. Under the conditions of glucose starvation, activation of the cAMP pathway causes Sp1 protein deglycosylation, resulting in a decreased Sp1 binding activity and an increased susceptibility to proteasome-dependent degradation (28). By contrast, hyperglycemia and elevated glucosamine cause Sp1 protein hyperglycosylation and increase Sp1-mediated gene transcription (28, 29). In vitro O-GlcNAcylation of the transactivation domain of Sp1 protein inhibits its interaction with TATA-binding protein-associated factor, TAFI 10 or holo-Sp1 (31). The role of Sp1 glycosylation in LPS signaling process has not been studied. We showed here that in vitro deglycosylation of Sp1 protein in control NP reduced Sp1 binding activity, indicating that glycosylation of Sp1 protein is required for its DNA binding function. Our result is consistent with a previous report demonstrating that deglycosylation of Sp1 protein reduced its DNA binding activity (28). However, the change in glycosylation state of Sp1 protein is unlikely to play a role in LPS signaling, since we showed here that LPS did not change the Sp1 glycosylation state.

We showed that LPS reduced Sp1 protein level, but not Sp1 mRNA abundance, indicating that reduction in the Sp1 protein level induced by LPS did not result from a reduced Sp1 mRNA expression. Our data cannot role out the possibility that LPS may decrease Sp1 protein translation. It is not possible to test protein synthesis inhibitor on real life animals. However, we detected two smaller peptides immunoreactive to Sp1 antibody on our Western blot membrane, and these smaller peptides were seen only in nuclear proteins from lungs challenged with LPS for 60 and 240 min but not in nuclear protein from other groups of lungs. This is consistent with our observation that nuclear proteins from lungs challenged with LPS for 60, 120, and 240 min showed a dramatically reduced Sp1 protein level. Thus, it is more likely that LPS down-regulates Sp1 binding activity through promoting Sp1 protein degradation. In agreement with our conclusion, several groups have demonstrated that stimulation of cells with various stimuli reduced Sp1 binding activity by accelerating Sp1 protein degradation, although the proteases that degrade SP1 protein appear to vary with cell type and stimuli (26, 28, 32, 33). The protease responsible for LPS-induced Sp1 protein degradation remains to be determined. Investigation is under the way to identify and isolate the Sp1-degrading enzyme.

We detected two Sp1 mRNA transcripts in both control and LPS-challenged lungs. Our observation is consistent with the early report that human Sp1 mRNA from HeLa cells showed two transcripts (8.2 and 4.1 bp, respectively) (41). Because there is only one mouse Sp1 gene located on chromosome 15 (43), these two transcripts are likely to be alternative splicing products.

Prior studies on the pathophysiology of sepsis have exclusively focused on the induction of proinflammatory genes and the roles of these gene products in the inflammatory response. We demonstrated here that challenge of mice with LPS in vivo resulted in a significant down-regulation of Sp1 binding activity, Sp1 protein, and Sp1-regulated genes. Sp1 plays a critical role in the transcription of very large number of genes, mainly housekeeping genes, tissue-specific genes, and cell cycle-regulated genes (19-21). This raises the intriguing question of whether LPS could cause an inflammatory suppression of normal physiological functions through down-regulation of Sp1 protein. A suppressed or diminished Sp1 activity can lead to a reduced transcription of a large number of housekeeping genes and reduced production those gene products required for the regulation of many physiological functions and for the maintenance of homeostasis. This can disrupt normal physiological processes and promote multiple organ dysfunctions.

In summary, we demonstrated that challenge of mice with LPS dramatically down-regulated the Sp1 DNA binding activity in the lungs, which was accompanied by and temporally correlated to the down-regulation of eNOS and COX-1 mRNA expression, two most important Sp1-dependent genes. LPS dephosphorylated Sp1 protein at serine and threonine residues but not at a tyrosine residue. In vitro dephosphorylation of Sp1 protein significantly reduced its DNA binding activity. Removal of the O-linked N-acetylglucosamine from control Sp1 protein also reduced Sp1 binding activity. However, LPS did not cause Sp1 protein deglycosylation. LPS challenge significantly reduced nuclear Sp1 protein level but had no effect on Sp1 mRNA abundance and on Sp1 protein nuclear translocation. Both Sp1 protein dephosphorylation and degradation are temporally correlated to the reduced Sp1 binding activity. These results indicate that LPS down-regulates Sp1 DNA binding activity in vivo through promoting Sp1 protein dephosphorylation and degradation.

	FOOTNOTES

^* This work was supported in part by the North Shore LIJ Research Institute Faculty Award Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Division of Pulmonary and Critical Care Medicine (RM C-20), Long Island Jewish Medical Center, New Hyde Park, NY 11040. Tel.: 718-470-7253; Fax: 718-470-1507; E-mail: Sliu@lij.edu.

Published, JBC Papers in Press, June 27, 2002, DOI 10.1074/jbc.M205544200

	ABBREVIATIONS

The abbreviations used are: LPS, lipopolysaccharide; O-GlcNAc, O-linked N-acetylglucosamine; CIP, calf intestinal alkaline phosphatase; NAG, -N-acetylglucosaminidase; EMSA, electrophoretic mobility shift assay; NP, nuclear protein; eNOS, endothelial nitric-oxide synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; COX-1, cyclooxygenase-1.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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